This Proposal will study the hypothesis that pharmacodynamic studies of whole extracts of tumor specimens of patients treated with 5-FU plus pure (/)-leucovorin will be improved by our newly-developed methods for quantitative histopathology. Our biochemical data suggest that the goals of 5-FU/leucovorin therapy are decreases in intratumoral thymidylate synthase (TS) and expansion of H4PteGlu pools. Tumors of patients with breast and gastrointestinal cancer will be biopsied before and after 5- FU(/)-leucovorin therapy and at tumor progression. Equal hemi-cylinder tumor biopsies will be taken for surgical pathology and for tissue homogenization assays of TS, tetrahydrofolate, 5,10- methylenetetrahydrofolate, dUMP, and FdUMP, by established [3H]FdUMP ligand-binding assays, and for TS mRNA by the Danenberg assay. TS protein will also be quantitated by ELISA assay using a rabbit polyclonal anti- human TS antibody preparation. Pathology studies of 4-micron sections will include immunoperoxidase antibody staining for TS, nuclear staining for cell counts, common-leukocyte-antigen staining for white cells, and Ki 67 staining for S-phase and growth fraction determination. Initial studies will include comparison of fresh frozen vs. paraffin-embedded tissues. The antibodies used for TS immunostaining may also enable visualization of H4PteGlun and 5,10-CH2-H4PteGlun pools, by forming TS-FdUMP-folate ternary complexes in situ, using exogenous TS addition. Quantitation of the numbers, intensities, and areas of immunostaining will be done by digital image analysis using video microscopy and JAVA software; a novel method for validation of this approach will sue common-embedding of reference cells in the specimen blocks for same-frame calibration of parameters. Double- labeling, such as immunofluorescence combined with immunoperoxidase staining for TS will permit quantitation of TS antigen levels by cell type. In addition, the image analysis system should be useful for quantitation of cells visualized by in situ mRNA hybridization of transcripts of interest.